Carotenoids are pigments that are ubiquitous throughout nature and synthesized by all photosynthetic organisms, and in some heterotrophic growing bacteria and fungi. Carotenoids provide color for flowers, vegetables, insects, fish and birds. Colors of carotenoid range from yellow to red with variations of brown and purple. As precursors of vitamin A, carotenoids are fundamental components in our diet and they play additional important role in human health. Industrial uses of carotenoids include pharmaceuticals, food supplements, animal feed additives and colorants in cosmetics to mention a few.
Because animals are unable to synthesize carotenoids de novo, they must obtain them by dietary means. Thus, manipulation of carotenoid production and composition in plants or bacteria can provide new or improved source for carotenoids.
Carotenoids come in many different forms and chemical structures. Most naturally occurring carotenoids are hydrophobic tetraterpenoids containing a C40 methyl-branched hydrocarbon backbone derived from successive condensation of eight C5 isoprene units (IPP). In addition, rare carotenoids with longer or shorter backbones occur in some species of nonphotosynthetic bacteria. The term “carotenoid” actually include both carotenes and xanthophylls. A “carotene” refers to a hydrocarbon carotenoid. Carotene derivatives that contain one or more oxygen atoms, in the form of hydroxy-, methoxy-, oxo-, epoxy-, carboxy-, or aldehydic functional groups, or within glycosides, glycoside esters, or sulfates, are collectively known as “xanthophylls”. Carotenoids are furthermore described as being acyclic, monocyclic, or bicyclic depending on whether the ends of the hydrocarbon backbones have been cyclized to yield aliphatic or cyclic ring structures (G. Armstrong, (1999) In Comprehensive Natural Products Chemistry, Elsevier Press, volume 2, pp 321–352).
Carotenoid biosynthesis starts with the isoprenoid pathway and the generation of a C5 isoprene unit, isopentenyl pyrophosphate (IPP). IPP is condensed with its isomer dimethylallyl pyrophophate (DMAPP) to form the C10, geranyl pyrophosphate (GPP), and elongated to the C15, farnesyl pyrophosphate (FPP). FPP synthesis is common to both carotenogenic and non-carotenogenic bacteria. Enzymes in subsequent carotenoid pathways generate carotenoid pigments from the FPP precursor and can be divided into two categories: carotene backbone synthesis enzymes and subsequent modification enzymes. The backbone synthesis enzymes include geranyl geranyl pyrophosphate synthase, phytoene synthase, phytoene dehydrogenase and lycopene cyclase, etc. The modification enzymes include ketolases, hydroxylases, dehydratases, glycosylases, etc. Unlike genes in the upstream isoprenoid pathway that are common in all organisms, the downstream carotenoid modifying enzymes are less common.
Carotenoid hydroxylases are a class of enzymes that introduce hydroxyl groups to the ionone ring of the cyclic carotenoids, such as β-carotene, echinenone, 3′-hydroxyechinenone, β-cryptoxanthin, adonirubin, and canthaxanthin to produce hydroxylated carotenoids. Examples of such carotenoids include astaxanthin, β-cryptoxanthin, zeaxanthin, 3-hydroxyechinenone, 3′-hydroxyechinenone, adonirubin, adonixanthin, tetrhydroxy-β,β′-caroten-4,4′-dione, tetrahydroxy-β,β′-caroten-4-one, caloxanthin, erythroxanthin, nostoxanthin, flexixanthin, 3-hydroxy-γ-carotene, 3-hydroxy-4-keto-γ-carotene, bacteriorubixanthin, bacteriorubixanthinal, and lutein.
Several classes of carotenoid hydroxylases have been reported (i.e. CrtR-type and CrtZ-type). Both CrtR and CrtZ enzymes catalyze addition of hydroxyl groups to the β-ionone rings of cyclic carotenoids. However, no significant sequence homology exists between CrtR hydroxylases and the CrtZ hydroxylases. The CrtR-type carotenoid hydroxylases have been reported in Cyanobacteria such as Synechocystis sp. PCC 6803 (Lagarde, D., and Vermaas, W., FEBS Lett., 454(3):247–251 (1999) and in plants. The CrtZ-type carotenoid hydroxylases have been reported from a variety of bacterial, fungal, algal, and plant species. Examples include, but are not limited to, bacterial species such as Pantoea stewartii (WO 03/016503; WO 02/079395), Erwinia uredovora (EP 393690 B1; Misawa et al., J. Bacteriol., 172(12):6704–6712 (1990)), Erwinia herbicola (Hundle et al., Mol. Gen Genet., 245(4):406–416 (1994); Hundle et al., FEBS Lett. 315(3):329–334 (1993); Schnurr et al., FEMS Microbiol. Lett., 78(2–3):157–161 (1991); and U.S. Pat. No. 5,684,238), Agrobacterium aurantiacum (Misawa et al., J. Bacteriol., 177(22):6575–6584 (1995); U.S. Pat. No. 5,811,273), Alcaligenes sp. (U.S. Pat. No. 5,811,273), Flavobacterium sp. (U.S. Pat. No. 6,677,134; U.S. Pat. No. 6,291,204; US 2002147371; WO 2004029275; and Pasamontes et al., Gene, 185(1):35–41 (1997)), Paracoccus sp. (CN 1380415), Haematococcus pluvialis (WO 00/061764; Linden, H., Biochimica et Biophysica Acta, 1446(3):203–212 (1999)), Brevundimonas vesicularis DC263 (U.S. Ser. No. 60/601,947), Enterobacteriaceae strain DC260 (U.S. Ser. No. 10/808,979, and plant species such as Arabidopsis thaliana (Tian, L. and DellaPenna, D., Plant Mol. Biol., 47(3):379–388 (2001); US 2002102631).
Carotenoid ketolases are enzymes that introduce keto groups to the β-ionone ring of the cyclic carotenoids, such as β-carotene, echinenone, β-cryptoxanthin, adonixanthin, 3′-hydroxyechinenone, 3-hydroxyechinenone, and zeaxanthin to produce ketocarotenoids. Examples of ketocarotenoids include, but are not limited to astaxanthin, canthaxanthin, adonixanthin, adonirubin, echinenone, 3-hydroxyechinenone, 3′-hydroxyechinenone, 4-keto-gamma-carotene, 4-keto-rubixanthin, 4-keto-torulene, 3-hydroxy-4-keto-torulene, deoxyflexixanthin, and myxobactone.
Several classes of carotenoid ketolases have been reported (Hannibal et al., J. Bacteriol., 182: 3850–3853 (2000)). These include CrtW ketolases from Agrobacterium aurantiacum (Misawa et al., J. Bacteriol., 177(22):6575–6584 (1995); WO 99/07867), Bradyrhizobium sp. ORS278 (Hannibal et al., J. Bacteriol., 182(13):3850–3853 (2000)), Brevundimonas aurantiaca (De Souza et al., WO 02/79395), Paracoccus marcusii (Yao et al., CN1380415); Bkt ketolases from Haematococcus pluvialis (Sun et al., Proc. Natl. Acad. Sci. USA, 95(19):11482–11488 (1998); Linde, H. and Sandmann, G., EP1173579; Breitenbach et al., FEMS Microbiol. Lett., 404(2–3):241–246 (1996)); and CrtO ketolases from Synechocystis sp. (Lagarde et al., Appl. Environ. Microbiol., 66(1):64–72 (2000); Masamoto et al., Plant Cell Physiol., 39(5):560–564 (2000); FR 2792335; Cheng et al., WO 03/012056 corresponding to U.S. Ser. No. 10/209,372)), Rhodococcus erythropolis (Cheng et al., supra), Deinococcus radiodurans (Cheng et al., supra), and Gloeobacter violaceus (Nakamura et al., DNA Res., 10:181–201 (2003)). It should be noted that the CrtO ketolase reported in Haematococcus pluvialis (Harker, M. and Hirschberg, J., FEBS Lett., 404(2–3):129–134 (1997); U.S. Pat. No. 5,965,795; U.S. Pat. No. 5,916,791; and U.S. Pat. No. 6,218,599) appears to be a CrtW/Bkt-type ketolase based on the size (nucleotide coding sequence length <1000 bp) and homology to other CrtW/Bkt ketolases. Bkt ketolases appear to be closely related to CrtW ketolases, sharing very little structural similarity to the CrtO ketolases based on nucleotide and amino acid sequence comparisons (Cheng, et al, supra). For example, a search of the publicly available sequences using the Haematococcus pluvialis Bkt ketolase sequence returned matches that most closely matched other CrtW-type ketolases. CrtW/Bkt ketolases are generally encoded by nucleic acid fragments about 800–1000 bp in length, while CrtO ketolases are normally encoded by a nucleic acid fragments of about 1.6 kb in size. Cheng et al. defines CrtO ketolases based on the existence of six conserved motifs considered diagnostic for all CrtO ketolases. The reported CrtO ketolases from Rhodococcus erythropolis, Deinococcus radiodurans, and Synechocystis sp. PCC6803 are comprised of these diagnostic motifs (U.S. Ser. No. 10/209,372).
The wildtype CrtO ketolases reported by Cheng et al. generally exhibit much lower activity when producing ketocarotenoids (i.e. canthaxanthin) from β-carotene in comparison to the reported CrtW ketolases (U.S. Ser. No. 10/209,372). U.S. Ser. No. 10/209,372 reports that, the use of recombinatly expressed R. erythropolis AN12 CrtO ketolase resulted in only 30% conversion of the initial substrate (β-carotene) into canthaxanthin (35% of the initial β-carotene was converted to echinenone with the remaining 35% remaining as β-carotene).
For biosynthesis of astaxanthin, a carotenoid ketolase and a carotenoid hydroxylase have to interact efficiently (Steiger, S. and Sandmann, G., Biotechnol Lett., 26:813–817 (2004)). As shown in FIG. 1, many carotenoid ketolases and carotenoid hydroxylases exhibit some level of substrate flexibility. This leads to a variety of possible enzymatic reactions (producing various intermediates from β-carotene) that may be necessary to produce astaxanthin. Depending upon the activity and substrate specificity of both the ketolase and hydroxylase used, it is often difficult to predict those combinations that will result in optimal production of astaxanthin. For example, it has been reported that hydroxylases from cyanobacteria are not able to accept echinenone or canthaxanthin as substrates for hydroxylation. Conversely, certain ketolases have been reported to be unable to use hydroxylated carotenoids, such as zeaxanthin, as suitable substrates (Steiger and Sandmann, supra). Coexpression of a carotenoid ketolase and a carotenoid hydroxylase that are able to efficiently work together is crucial for producing substantial amounts of astaxathin in a recombinant host cell. When using a recombinant host cell capable of producing suitable amounts of β-carotene, one must take into account a variety of variables that factor into optimal astaxanthin production including, but not limited to 1) substrate flexibility associated with each ketolase and the hydroxylase used, 2) the ability of each enzyme to efficiently hydroxylate/ketolate one or more of the possible carotenoid substrates, and 3) the relative balance of ketolase and hydroxylase enzymatic activity. For example, one can invision a scenario where a ketolase, which selectively uses β-carotene as a substrate, should not be coexpressed with a hydroxylase having much higher activity for β-carotene. In such an instance, the majority of the β-carotene would be expected to be converted into hydroxylated carotenoids (such as zeaxanthin) that may not be recognized by the ketolase, resulting in less than optimal production of astaxanthin.
The CrtO ketolase from R. erythropolis AN12 has been protein engineered for increased canthaxanthin production (U.S. Ser. No. 60/577,970). Coexpression of the CrtO ketolase with a CrtZ cartenoid hydroxylase was expected to result in the production of astaxanthin (FIG. 1). However, as described in the present disclosure, coexpression of the best canthaxanthin producing CrtO ketolase mutant (“320SHU001” herein referred to as “crtO-SHU0001”) from U.S. Ser. No. 60/577,970 with several different CrtZ hydroxylases did not result in the expected production of astaxanthin.
The problem to be solved is to provide nucleic acid molecules encoding at least one CrtO ketolase and at least one CrtZ hydroxylase that can efficiently work together to produce astaxanthin in a recombinant host cell. A further problem to be solved is to provide a method to produce matched pairs of carotenoid ketolases and carotenoid hydroxylases having astaxanthin biosynthesis activity.
The stated problem has been solved by providing several CrtO ketolase/CrtZ hydroxylase mutants exhibiting improved astaxanthin production in the context of a carotenoid biosynthetic pathway. A nucleic acid fragment (comprised of crtOZ genes) encoding enzymes incapable of producing more than trace amounts astaxanthin was protein engineered using errror-prone PCR to create several CrtO/Z combinations having the ability to produce significant amounts of astaxanthin when expressed in a recombinant host cell. Methods to produce and/or alter astaxanthin production in recombinant host cells using the present genes are also provided.
Additionally, a method to produce combinations of carotenoid ketolases and carotenoid hydroxylases having improved astaxanthin biosynthesis activity is also provided. The method is comprised of simultaneously mutating nucleic acid fragments encoding one or more carotenoid ketolases and one or more carotenoid hydroxylases and screening recombinants for improvements in astaxanthin production.